Sustained release ionic conjugate

ABSTRACT

A method of spherifying a sustained release ionic conjugate which contains a free carboxyl group-containing biodegradable polymer and a free amino group-containing drug which are ionically bonded to each other.

TECHNICAL FIELD

This invention relates to sustained release drug delivery systems and,in particular, to a method of making microparticles of a sustainedrelease ionic conjugate.

BACKGROUND ART

Biodegradable polymeric drug delivery formulations have been developedand utilized for the controlled in vivo release of drugs. See, e.g.,U.S. Pat. Nos. 3,773,919 and 4,767,628. Such biodegradable polymericformulations are designed to allow an entrapped drug to slowly diffusethrough a polymer matrix or coating when the biodegradable polymer isdepolymerized.

International Publication No. WO 94/15587 describes sustained releaseionic molecular conjugates of polyesters and drugs. Since polyesterdegradation is a key step in the release process, the surface area ofthe conjugate particles can control the release profile of the drug fromthe conjugate. Thus, the conjugate particles should be of similar sizeand shape to insure both the minimum and reproducible surface area,e.g., microspheres.

DISCLOSURE OF INVENTION

In one aspect, this invention features a method of making microparticlesof a sustained release ionic conjugate containing a free carboxylgroup-containing biodegradable polymer (a polyester made of monomerssuch as lactic acid, e-caprolic acid, glycolic acid, trimethylenecarbonate, or p-dioxanone; or a copolymer thereof, the monomers can beoptically isomers or racemates) and a free amino group-containing drug(e.g., a peptide drug such as somatostatin or LHRH) which are ionicallybonded to each other. The method includes the steps of (1) obtaining afirst solution in which the conjugate is dissolved; (2) mixing the firstsolution (added as small droplets, e.g., through an atomizing nozzlesuch as sonic nozzle, pneumatic nozzle, rotary atomizer, or pressurenozzle) with a first liquid to form a first dispersion, wherein thefirst liquid is miscible with the first solution, and the conjugate isnot soluble in the first liquid and precipitates out of the firstdispersion; and (3) isolating the conjugate from the first dispersion.

In one embodiment, the drug is soluble in the first liquid, which can bean alcohol (e.g., ethanol or isopropyl alcohol), hexane, or water; or amixture thereof. When ethanol is used as the first liquid, it can bemaintained between about 0° C. and −30° C. when it is being used. Whenisopropyl alcohol is used, it can be maintained between about 0° C. and−70° C., e.g., cooled by the addition of dry ice.

The first solution, which may contain acetone, dichloromethane,acetonitrile, ethyl acetate, tetrahydrofuran, or glyme, or mixturesthereof can be obtained by (1) dissolving the biodegradable polymer in asecond liquid (e.g., acetone, tetrahydrofuran, glycone, ethyl acetate,methyl acetate, acetonitrile, ethyl formate, or glyme; or a mixturethereof) to form a second solution; (2) dissolving the drug in a thirdliquid (e.g., water or acetone; or a mixture thereof) to form a thirdsolution, wherein the third liquid is miscible with the first liquid andthe second liquid; and (3) mixing the second solution and the thirdsolution to form the first solution, wherein the mixing causes the drugto ionically bond to the biodegradable polymer and form the conjugate inthe first solution. The first solution may comprise up to 40% by weightof the conjugate (e.g., between 25 and 35 percent by weight of theconjugate). In one example, a base, e.g., NaOH or KOH, can be added tothe second solution prior to mixing the second solution and the thirdsolution. Neutralization of the carboxyl groups of the biodegradablepolymer with the base facilitates the formation of the ionic conjugate.

Alternatively, the first solution is obtained by dissolving thebiodegradable polymer and the drug in a second liquid (e.g., acetone ora mixture of acetone and water) to form the first solution, therebyforming the conjugate in the first solution. According to this method,the biodegradable polymer can be first dissolved in the second liquid, abase is then added to the second solution, and the drug is subsequentlydissolved in the second liquid. Also, if desired, the first solution canbe partially or completely evaporated from the first dispersion prior toisolation of the conjugate. The processed conjugate can be convenientlyisolated by centrifuging or filtering the first dispersion, and theisolated conjugate can be mixed with an aqueous mannitol solution priorto vacuum drying (e.g., lyophilization). The isolated conjugate can befurther shaped into a film or a rod. The isolated conjugate can also bespherified into microspheres of average diameter of 5 to 200 μm, e.g.,as described herein. By “spherification” or “spherifying” is meant theprocessing of a microparticle into a shape close to a sphere.

In another aspect, this invention features a method of spherifying asustained release ionic conjugate as described above. The methodincludes the steps of (1) mixing the conjugate with a first liquid(e.g., an oil such as silicon oil, mineral oil, sesame oil, or avegetable oil) to form a first dispersion, wherein the conjugate has theshape of a microparticle and is not soluble in the first liquid; (2)heating the first dispersion to a temperature greater than the Tg or Tmof the conjugate; (3) cooling the first dispersion below the Tg or Tm ofthe conjugate; (4) mixing the first dispersion with a second liquid(e.g., hexane, heptane, isopropyl myristate, or an alcohol such asethanol or isopropyl alcohol) to form a second dispersion, wherein thesecond liquid is miscible with the first liquid and the conjugate is notsoluble in the second liquid; and (5) isolating the conjugate from thesecond dispersion. The conjugate may have the shape of a microcapsulewith an average diameter of between 5 μm to 200 μm prior to mixing withthe first liquid, and the first dispersion thus formed is vigorouslystirred while being heated to aid in the separation of the particles.Once the conjugate has been isolated, it can be rinsed with the secondliquid and then vacuum dried. Optionally, it can also be mixed with anaqueous mannitol solution prior to vacuum drying.

A third aspect of this invention features a method of spherifying theabove-described sustained release ionic conjugate (e.g., a microcapsulehaving an average diameter of between 5 μm to 200 μm). The methodincludes the steps of (1) mixing the conjugate in a first liquid (e.g.,water) to form a first dispersion, wherein the conjugate is in the shapeof a microparticle and the conjugate is not soluble in the first liquid;(2) stirring the first dispersion; (3) mixing the stirred dispersionwith a second liquid (e.g., dichloromethane or chloroform) in such anamount so that it is absorbed by the conjugate but does not solubilizethe conjugate, wherein the second liquid is miscible with the firstliquid; (4) evaporating the second liquid from the first dispersion; and(5) isolating the precipitated conjugate from the first dispersion. Ifnecessary, the method may further include the step of adding asurfactant (e.g., lecithin, Tween 20, polysorbate, or lauryl sulfate) tothe first dispersion to aid in the stabilization of the firstdispersion, and the isolated conjugate can be rinsed with the firstliquid and vacuum dried. Again, the isolated conjugate can be mixed withan aqueous mannitol solution prior to vacuum drying.

In a further aspect of this invention, this invention features a methodof spherifying the above-described sustained release ionic conjugate.The method includes the steps of (1) dissolving the conjugate in a firstliquid (e.g., acetonitrile) to form a first solution; (2) stirring thefirst solution with a second liquid (e.g., an oil) to form a firstdispersion, wherein the second liquid is immiscible with the firstsolution; (3) evaporating the first liquid from the first dispersion toprecipitate the conjugate from the first dispersion; and (4) isolatingthe precipitate conjugate from the first dispersion. In the stirringstep, the first solution can be added to the second liquid as smalldroplets.

The above method can further include the step of rinsing the isolatedconjugate with a third liquid (e.g., hexane, heptane, or octane) whichis miscible with the second liquid and not a solvent for the isolatedconjugate. If desired, the isolated conjugate can be mixed with anaqueous mannitol solution prior to vacuum drying.

The biodegradable polymer in the above-described conjugate may containat least one free carboxyl group (e.g., two to ten free carboxyl groupsper polymer chain). Examples of carboxylic acid containing biodegradablepolymers include polyesters containing units of lactic acid, e-caprolicacid, p-dioxanone, e-caprionic acid, substituted and unsubstitutedtrimethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxepan-2-one, glycolicacid, alkylene oxylate, cycloalkylene, cycloalkylene oxylate, alkylenesuccinate, or 3-hydroxy butyrate in optically active forms or asracemates; or copolymers of any of the above. Additional free carboxylicacid groups can be incorporated into the biodegradable polyester byreaction, e.g., ring opening polymerization or polycondensation, withpolycarboxylic acids such as malic acid, tartaric acid, pamoic acid,citric acid, succinic anhydride, and glutaric anhydride. Thus, thebiodegradable polymer can be a water insoluble polyester includinglactic acid units with or without glycolic acid units. Otherbiodegradable polymers such as polyorthoesters, polyorthocarbonates, andpolyantals may also be used. The biodegradable polymer may have anaverage degree of polymerization, e.g., average number of monomers perpolymer chain, between 10 and 300.

The drug has one or more (e.g., one to ten) free amine groups. In oneembodiment, the drug is an acid-stable peptide. Examples of suitableacid-stable peptides include growth hormone releasing peptide (GHRP),luteinizing hormone-releasing hormone (LHRH), adrenomedullin, growthhormone, somatostatin, bombesin, gastrin releasing peptide (GRP),calcitonin, bradykinin, galanin, melanocyte stimulating hormone (MSH),growth hormone releasing factor (GRF), amylin, adrenomedullin,tachykinins, secretin, parathyroid hormone (PTH), erkephalin,endothelin, calcitonin gene releasing peptide (CGRP), neuromedins,parathyroid hormone related protein (PTHrP), glucagon, neurotensin,adrenocorticotrophic hormone (ACTH), peptide YY (PYY), glucagonreleasing peptide (GLP), vasoactive intestinal peptide (VIP), pituitaryadenylated cyclase activating peptide (PACAP), motilin, substance P,neuropeptide Y (NPY), TSH, and analogs and fragments thereof. The drugmay be soluble (e.g., greater than 0.1 mg/ml; preferably, greater than1.0 mg/ml) in the first liquid.

Other features and advantages of the present invention will be apparentfrom the detailed description and from the claims.

It is believed that one skilled in the art can, based on the descriptionherein, utilize the present invention to its fullest extent. Thefollowing specific embodiments are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference.

BEST MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

18.0 g of a 6,000 g/mol 66/32/2 poly-L-lactic-co-glycolic-co-D,L-malicacid copolymer (66 percent L-lactic acid, 32 percent glycolic, and 2percent malic acid; acid number of 0.373 milliequivalents/g) wasdissolved in 180 g of acetone (solution of 10% copolymer by weight).14.4 ml of 0.5 N aqueous NaOH was added to form the sodium carboxylateof the polymer. 4.28 g of the acetate salt of the peptide Lanreotide™(Kinerton, Dublin, Ireland; D-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-NH₂;acetate content=9.60 percent by weight) was separately dissolved in amixture of 10 g of acetone and 10 g of deionized water. The amount ofpeptide dissolved corresponded to the stoichiometric ratio of acidgroups from the copolymer (e.g., one) and the free amino groups for thepeptide (e.g., two). The peptide solution was then added dropwise to thecopolymer solution, and the resulting solution was stirred for two hoursto allow for salt exchange and the resulting formation of thepolymer/peptide ionic conjugate (PPIC).

EXAMPLE 2

In a temperature controlled jacketed reactor (Schott Glass AGB, Dublin,Ireland), a two liter bath of deionized water was precooled to 0° C. andwas vigorously stirred. The above PPIC solution of Example 1 was thenslowly added to the reactor using a Masterflex pump (BioblockScientific, Illkvch, France) which produced a flow rate of 10-15 ml/minthrough a silicone tubing fitted with a 19 gauge needle at its tip. ThePPIC solution was fed through the needle that was positioned above awater bath at 0° C. The PPIC precipitated in the bath as small, solidparticles. The solid particles were then separated from the supernatantby centrifugation (30 minutes at 5000 rpm and 0-5° C.), rinsed withfresh deionized water, resuspended water, recentrifuged, and thenlyophilized. The isolated conjugate was filtered through a 100 μm sieveto remove any large particles which would not be capable of beinginjected through a 21 gauge needle. An analysis of the resultingparticle sizes is described in Table 1.

EXAMPLE 3

The PPIC solution of Example 1 was also precipitated as described abovein Example 2, except that a bath of ethanol at a temperature of −20° C.was used instead of a bath of water at 0° C. An analysis of theresulting particle sizes is described in Table I.

EXAMPLE 4

The PPIC solution of Example 1 was also dispersed at a controlled flowrate of 4 ml/min through an atomizing nozzle containing a hollow tip(Bioblock; 50 Watts, 20 kHz) over a bath of ethanol at −10° C. in atemperature controlled jacket reactor. In this nebulization process, thecopolymer solution was released from the probe as a fine mist of smalldroplets. The small droplets fell into the ethanol bath, causing thedeionized water and acetone to be extracted from the droplets. As aresult, the copolymer droplets hardened as small, solid particles. Theparticles were then recovered by centrifugation and lyophilized. Ananalysis of the resulting particle sizes is listed in Table I. What ismeant by Diameter −10 (i.e., D0.1), Diameter −50.(i.e., D0.5), orDiameter −90 (i.e., D0.9) is the smallest diameter which is greater than10%, 50%, and 90% of the total particles, respectively. What is meant byspecific area is the average specific area of the resulting particles.TABLE I Specific Diameter-10 Diameter-50 Diameter-90 Area Example (μm)(μm) (μm) (m²/g) 2 10 30 62 18.64 3 9 37 89 6.42 4 13 46 95 22.61

EXAMPLE 5

5.0 g of the PPIC described above in Example 4 was dissolved in 20 g ofacetone (concentration of 20% PPIC by weight). This solution was thennebulized at a flow rate of 4.0 ml/min over 500 ml of bath of ethanol at−10° C. as described in Example 4. After preparation of the PPICparticles in the bath, 500 ml of deionized water was added to the bath,and the bath was then brought up to 0° C. The bath was then stirred for30 min, brought up to 20° C., and stirred for an additional 30 min. ThePPIC particles were then recovered by filtration and dried under vacuumat room temperature. An analysis of the resulting particles is listed inTable II. TABLE II Specific D 0.1 D 0.5 D 0.9 Area Example (μm) (μm)(μm) (m²/g) #4 13 46 95 22.61 #5 50 99 180 0.11

As shown in Table II, different morphologies of particles were obtained.The particles of Example 4 were larger and had a lower specific area. Asindicated by electron scanning microscope, the particles obtained inExample 4 were also more porous, likely because of the frozen waterwhich remained in the particles upon precipitation. When the bathdispersion was returned to room temperature, the water thawed and flowedinto the ethanol bath, leaving open channels in the microparticles.Consequently, these particles were more brittle and generated fragmentsof small size.

EXAMPLE 6

A solution of PPIC described above in Example 5 was nebulized at 2.5ml/min over 1.5 liters of deionized water at 0° C. An analysis of theresulting particle sizes is listed in Table III.

EXAMPLE 7

A solution of PPIC described above in Example 5 was nebulized at 2.5ml/min over 1.5 liters of ethanol at −10° C. An analysis of theresulting particle sizes is listed in Table III. TABLE III Specific D0.1 D 0.5 D 0.9 Area Example (μm) (μm) (μm) (m²/g) 6 53.4 154.3 329.1n/a 7 42.4 87.2 170.1 0.20

EXAMPLE 8

Two solutions of PPIC are prepared in acetone as described above inExample 5. The first solution has a PPIC concentration of 15% while thesecond solution has a PPIC concentration of 20%. The solutions arenebulized over a bath of ethanol at −10° C. at flow rates of 2.5, 3.5,and 5.0 ml/min as described in Example 5. An analysis of the resultingparticle size is listed in Table IV. TABLE IV Specific Feeding Rate D0.1 D 0.5 D 0.9 Area Concentration (ml/min) (μm) (μm) (μm) (m²/g) 15%2.5 35.9 81.6 191.1 4.455 15% 3.5 34.4 80.2 188.3 8.336 15% 5.0 49.4163.6 397.8 n/a 20% 2.5 33.3 73.8 145.6 0.199 20% 3.5 50.8 112.7 241.90.579 20% 5.0 108.3 219.1 395.9 n/a

Analysis of the particles using scanning electron microscope revealedthat the particle size and the specific area increased with the increasein feeding rate.

EXAMPLE 9

5.0 g of PPIC microparticles of Example 4 was dissolved in 45 g ofacetone (concentration 10% by weight). The solution was then addeddropwise over a vigorously stirred 500 ml n-hexane at room temperature.The n-hexane solution turned cloudy as particles of PPIC precipitated.The PPIC was removed by filtration and dried under vacuum at roomtemperature.

EXAMPLE 10

In a jacketed reactor, 3.0 g of the PPIC microparticles described inExample 2 was dispersed in a vigorously stirred 250 ml of 12,500 csmedical grade silicon oil (Dow Coming, Midland, Mich.) (of 1% PPIC byweight). After the stirring, the mixture was then heated to 120° C.,which is above the Tg of 55° C. for the PPIC, and kept at thistemperature for 30 minutes. During this heating, the isolated individualparticles melted to fond spherical droplets. The dispersion was thencooled to 20° C. and then diluted with 1250 ml of hexane. Themicrospheres subsequently hardened, were recovered by filtration, wererinsed with fresh hexane, and were finally dried under vacuum. Thecharacteristics of the obtained microspheres are reported in Table V.The final microspheres had a small diameter as compared to those ofExample 2 as a result of the compaction of the particles during melting.TABLE V Specific D 0.1 D 0.5 D 0.9 Area Example (μm) (μm) (μm) (m²/g) #210 30 62 18.64 #7 2 10 47 <0.33

EXAMPLE 11

0.2 g of the PPIC microparticles described in Example 2 were dispersedin 5 ml of deionized water and vigorously stirred with a vortex shaker.100 ml microliters of dichloromethane (DCM) was then added over thestirred dispersion. The addition of a small amount of DCM caused aswelling of the surface of the PPIC particles. Stirring was kept at roomtemperature for 4 hours, allowing for evaporation of DCM andconsequential hardening of the swollen surface of the particles. Ascanning electron microscope showed that the resulting particles were ofspherical shape with a smoother surface as compared to the startingmaterial. Both the particle size distribution was narrowed, and themaximum particle size was reduced as a result of the increase in thedensity of the particles.

EXAMPLE 12

One liter of sesame seed oil (Vitamins, Inc., Chicago, Ill.) was placedin a 2 liter, three necked flask immersed in a water bath. The oil wasstirred at 600 rpm using a teflon stirring paddle connected to anoverhead stirring motor. 500 mg of the surfactant, soybean lecithin,(Sigma Chemicals, St. Louis, Mo.) was added to the sesame seed oil, andthe mixture was stirred for 10 min. 10 g of a PPIC formulation was thendissolved in 100 ml acetonitrile to give a clear solution. The PPICcompositions were made using Lanreotide™ conjugated with one of thefollowing three polymers: 64/34/2 poly-DL-lactic-co-glycolic-D,L-malicacid copolymer (M.W. avg 6,000) (Composition 1); 74/24/2poly-DL-lactic-co-glycolic-D,L-malic acid copolymer (M.W. avg 6,000)(Composition 2); and 98/2 poly-DL-lactic-co-D,L-malic acid copolymer(Composition 3).

This clear PPIC solution was added dropwise through a dropping funnel.When the addition was completed, the temperature of the external waterbath was raised to 40° C., and the oil was left stirring for 20 h. Oneliter of hexane was then added to dilute the sesame seed oil, and theoil was filtered through a medium fritted funnel. The microspherescollected in the filter funnel were further washed several times with500 ml in total volume of hexane. The particles are dried at 36° C. fortwo days under vacuum. Characteristics of the resulting microspheres arepresented in Table VI. TABLE VI Specific D 0.1 D 0.5 D 0.9 AreaComposition (μm) (μm) (μm) (m²/g) 1 13 28 57 0.1426 2 13 25 59 0.1395 314 25 51 0.1480

EXAMPLE 13

Reactor was loaded with monomers glycolide (Purac Biochem, Netherlands,84.83 g), lactide (PuracBiochem, Netherlands, 210.67 g) andL(+)-tartaric acid (Riedel-de Haen, Seelze, Germany, article number33,801, 4.50 g) and stannous 2-ethyl hexanoate (Sigma, St. Louis, Mo.,USA, article number S-3252) in toluene (Riedel-de Haen, Seelze, Germany)solution (0.1025 M, 4.34 ml). The L(+)-tartaric acid was previouslydried over phosphorous pentoxide (Riedel-de Haen, Seelze, Germany) in anAbderhalden drying apparatus for 10 hours. The reactor (connected topump via a liquid nitrogen trap) was then put under vacuum (0.04 mbar)with stirring for 50 minutes to remove toluene. The reactor, under anatmosphere of oxygen-free nitrogen (BOC gases, Dublin, freland, moisturecontent of 8 VPM), was then immersed in an oil bath (Temperature=200°C.) and stirring was increased to 125 rpm. Prior to immersion, a heatingtape (Thermolyne type 45500, input control setting=4) was placed on thereactor lid. The time taken to completely melt the reactor contents wasnoted, typically 10 minutes for a load of 300 g at 200° C. Samples weretaken every hour during synthesis and analyzed by GPC to determine thepercentage residual monomer and to obtain values for average molecularweight by number (Mn) and by weight (Mw). Typical reaction times are ofthe order of 6 hours.

An amorphous copolymer was obtained comprising 66.21% lactide units,33.11% glycolide units, and 0.68% tartaric acid units (66/33/1 PLGTA).The acid number of the titration was determined to 0.303milliequivalents/g (meq/g; the normality of NaOH multiplied by thevolume of NaOH solution required to neutralize one gram of polyester).The average number average molecular weight of the copolymer had a valueof 10,250, the average weight molecular weight of the copolymer was11,910 giving a Mw/Mn value of 1.16.

41.32 g of the above 10,000 g/mol 66/32/2poly-L-lactic-co-glycolic-co-L(+)-tartaric acid copolymer (acidnumber=0.303 meq/g) was dissolved in 165.52 g of acetone (Riedel-deHaen, Seelze, Germany) by sonication in a Branson sonication bath(Branson, Danbury, Conn., USA) to give a solution with a concentrationof 19.98% PLGTA by weight.

To this solution was added 37.6 ml 0.2 N sodium carbonate (Aldrich,Gillingham, Dorset, UK) thus providing a 1.2 times excess of sodium overcopolymer carboxyl groups. The solution was left to stir for 30 minutesto aid sodium salt formation. It was then fed to an atomizer nozzle at8.0 ml/min using a Masterflex pump (Cole Parmer, Barrington, Ill., USA).The solution was nebulized into a 6 L jacketed reactor containing 2 L ofde-ionized water cooled to 2.5° C. using a circulation bath (Huber,Offenburg, Germany). This water was stirred to 350 rpm using a 4-bladepaddle linked to a stirrer motor.

Once nebulization was complete, the dispersion was placed in 6centrifuge bottles and spun at 5000 rpm for 30 minutes in a Sorvallcentrifuge (DuPont Sorvall Products, Wilmington. Del., USA). Theresultant centrifuge cakes were resuspended in de-ionized water andre-spun. The supernatant was discarded and the cakes were frozen in afreezer overnight before being dried in a small-scale lyophilizer(Edwards, Crawley, West Sussex, UK) the next day. 33.16 g of washedcopolymer were recovered representing a yield of 80.24%.

4.92 g of the above 10,000 g/mol 66/33/1poly-L-lactic-co-glycolic-co-D,L-tartaric acid copolymer (66 percentL-lactic acid, 33 percent glycolic acid, and 1 percent tartaric acid)was dissolved in 11.58 g of acetonitrile (Ridel-de Haen, Seelze,Germany; HPLC grade) by sonication in a Branson sonication bath(Branson, Danbury, Conn., USA) and stirred on a stir-plate producing asolution with a concentration of 29.82% of PLGTA by weight.

This copolymer/acetonitrile solution was fed from a glass reservoirthrough an atomizer nozzle using an FMI revolving piston pump (FMI,Oyster Bay, N.Y., USA) set at 2.0 ml/min. The atomizer output power wasset at 50 W with an amplitude of 80%. The solution was nebulized into a6 L jacketed reactor containing 1.5 L of general purpose reagentisopropyl alcohol (Labscan, Dublin, Ireland), cooled to −70° C. by solidCO₂ pellets (AIG, Dublin, Ireland), and stirred at 300 rpm with a4-blade paddle linked to a stirrer motor. The temperature of theisopropyl alcohol remained at or close to −70° C. throughoutnebulization which lasted for approximately 8 minutes.

Once nebulization was complete, the dispersion was allowed to warm to10° C. of its own accord over a period of 5.5 hours. It was thenfiltered over a Whatman No. 1 filter paper (9 cm diameter) with the aidof a vacuum. The filter paper and cake were placed in a desiccator alongwith silica gel drying chips and a vacuum was pulled through anautomatic refrigeration trap at −110° C. After 24 hours 4.24 g ofmaterial were recovered. An analysis of the resulting particles islisted in Table VII. TABLE VII D 0.1 D 0.5 D 0.9 Specific Area Example #(μm) (μm) (μm) (μ²/g) 13 31 68 139 0.16

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the claims.

1. A method of making microparticles of a sustained release ionicconjugate containing a free carboxyl group-containing biodegradablepolymer and a free amino group-containing drug which are ionicallybonded to each other, the method comprising: obtaining a first solutionin which said conjugate is dissolved, wherein said solution comprisesacetone, acetonitrile, ethyl acetate, tetrahydrofuran, or glyme; mixingsaid first solution with a first liquid to form a first dispersion,wherein said first liquid is miscible with said first solution, and saidconjugate is not soluble in said first liquid and precipitates out ofsaid first dispersion; and isolating said conjugate from said firstdispersion.
 2. A method according to claim 1, wherein said firstsolution is added to said first liquid as small droplets.
 3. A methodaccording to claim 2, wherein said first solution is added to said firstliquid through an atomizing nozzle.
 4. A method according to claim 1,wherein said drug is a peptide.
 5. A method according to any claim 1,wherein said biodegradable polymer is a polyester made of lactic acid,e-caprolic acid, glycolic acid, trimethylene carbonate, or p-dioxanone;or a copolymer thereof.
 6. A method according to claim 1, wherein saiddrug is soluble in said first liquid.
 7. A method according to claim 1,wherein said biodegradable polymer is a polyester comprising lacticacid, or glycolic acid; or a copolymer thereof.
 8. A method according toclaim 7, wherein said polyester further contains malic acid, tartaricacid, citric acid, succinic acid, or glutaric acid.
 9. A methodaccording to claim 4, wherein said peptide is a somatostatin or LHRH.10. A method according to claim 1, wherein said first liquid is alcoholor water; or a mixture thereof.
 11. A method according to claim 10,wherein said first liquid is ethanol maintained between about 0° C. and−30° C. or isopropyl alcohol maintained between about 0° C. and −70° C.12. A method according to claim 1, wherein said first solution containsacetone or acetonitrile.
 13. A method according to claim 1, wherein saidfirst solution is obtained by: dissolving said biodegradable polymer ina second liquid to form a second solution; dissolving said drug in athird liquid to form a third solution, wherein said third liquid ismiscible with said first liquid and said second liquid; and mixing saidsecond solution and said third solution to form said first solution,wherein said mixing causes said drug to ionically bond to saidbiodegradable polymer and form said conjugate in said first solution.14. A method according to claim 13, wherein NaOH or KOH is added to thesecond solution prior to mixing said second solution and said thirdsolution.
 15. A method according to claim 13, wherein said second liquidis acetone; and said third liquid is water or acetone; or a mixturethereof.
 16. A method according to claim 1, wherein said first solutionis obtained by dissolving said biodegradable polymer and said drug in asecond liquid to form said first solution, thereby forming saidconjugate in said first solution.
 17. A method according to claim 16,wherein said second liquid is acetone or a mixture of acetone and water.18. A method according to claim 17, wherein said biodegradable polymeris first dissolved in said second liquid, a base is then added to saidsecond solution, and said drug is subsequently dissolved in said secondliquid.
 19. A method according to claim 1, wherein said conjugate isisolated by centrifuging or filtering said first dispersion.
 20. Amethod according to claim 19, wherein said first solution is partiallyor completely evaporated from said first dispersion prior to isolationof said conjugate.
 21. A method according to claim 20, wherein saidisolated conjugate is mixed with an aqueous mannitol solution prior tovacuum drying.
 22. A method of spherifying a sustained release ionicconjugate comprising a biodegradable polymer containing a free carboxylgroup containing biodegradable polymer and a free amino group-containingdrug which are ionically bonded to each other, said method comprising:mixing said conjugate with a first liquid to form a first dispersion,wherein said conjugate has the shape of a b microparticle and is notsoluble in said first liquid; heating said first dispersion to atemperature greater than the Tg or Tm of said conjugate; cooling saidfirst dispersion below the Tg or Tm of said conjugate; mixing said firstdispersion with a second liquid to form a second disperson, wherein saidsecond liquid is miscible with said first liquid and said conjugate isnot soluble in said second liquid: and isolating said conjugate fromsaid second dispersion.
 23. A method according to claim 22, wherein saidconjugate has the shape of a microcapsule which has an average diameterof between 5 μm to 200 μm prior to mixing with said first liquid andsaid first dispersion is stirred prior to said heating or cooling.
 24. Amethod according to claim 22, wherein said biodegradable polymer is apolyester made of lactic acid or glycolic acid; or a copolymer thereof.25. A method according to claim 22, wherein said drug is a peptide. 26.A method according to claim 22, wherein said first liquid is an oil andsaid second liquid is hexane.
 27. A method according to claim 22,further comprising: rinsing said isolated conjugate with said secondliquid; and vacuum drying said rinsed conjugate.
 28. A method accordingto claim 27, wherein said isolated conjugate is mixed with an aqueousmannitol solution prior to vacuum drying.
 29. A method of spherifying asustained release ionic conjugate containing a free carboxylgroup-containing biodegradable polymer and a free amino group-containingdrug which are ionically bonded to each other, said method comprising:dissolving said conjugate in a first liquid to form a first solution;stirring said first solution with a second liquid to form a firstdispersion, wherein said second liquid is immiscible with said firstsolution; evaporating said first liquid from said first dispersion toprecipitate said conjugate from said first dispersion; and isolatingsaid precipitate conjugate from said first dispersion.
 30. A methodaccording to claim 29, wherein said first solution is added to saidsecond liquid as small droplets.
 31. A method according to claim 29,wherein said first liquid is acetonitrile and said second liquid is anoil.
 32. A method according to claim 31, wherein said oil is siliconoil, mineral oil, sesame oil, or a vegetable oil.
 33. A method accordingto claim 29, wherein said biodegradable polymer is a polyestercomprising lactic acid or glycolic acid; or a copolymer thereof.
 34. Amethod according to claim 29, wherein said drug is a peptide.
 35. Amethod according to claim 29, further comprising rinsing said isolatedconjugate with a third liquid which is miscible with said second liquidand not a solvent for said isolated conjugate.
 36. A method according toclaim 35, wherein said third liquid is hexane, heptane, or octane.
 37. Amethod according to claim 29, wherein said isolated conjugate is mixedwith an aqueous mannitol solution prior to vacuum drying. 38-39.(cancelled)
 40. A method according to claim 8, wherein said polyestercomprises lactic acid, glycolic acid and tartaric acid. 41.Biodegradable polymer comprising lactic acid, ε-caproic acid, glycolicacid, trimethylene carbonate, p-dioxanone or a copolymer thereof andtartaric acid.
 42. Microparticles comprising a biodegradable polymeraccording to claim
 41. 43. Microparticles of a sustained release ionicconjugate comprising the biodegradable polymer according to claim 41 anda drug containing one or more free amino groups, wherein the polymer anddrug are ionically bonded.
 44. Microparticles according to claim 43wherein said drug is selected from the group consisting of growthhormone releasing peptide luteinizing hormone-releasing hormone,adrenomedullin, growth hormone, somatostatin, bombesin, gastrinreleasing peptide, calcitonin, bradykinin, galanin, melanocytestimulating hormone, growth hormone releasing factor, amylin,tachykinins, secretin, parathyroid hormone, enkephalin, endothelin,calcitonin gene releasing peptide, neuromedins, parathyroid hormonerelated protein, glucagon, neurotensin, adrenocorticotrophic hormone,peptide YY, glucagon releasing peptide, vasoactive intestinal peptide,pituitary adenylated cyclase activating peptide, motilin, substance P,neuropeptide Y and TSH or an analogue or a fragment thereof. 45.Microparticles according to claim 44 wherein said drug is somatostatinor LHRH or an analogue or a fragment thereof.
 46. Microparticlesaccording to claim 45 wherein said somatostatin analogue isD-β-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-Nh₂.
 47. The biodegradable polymeraccording to claim 41 comprising lactic acid glycolic acid and tartaricacid.
 48. Microparticles comprising a biodegradable polymer according toclaim
 47. 49. Microparticles of a sustained release ionic conjugatecomprising the biodegradable polymer according to claim 47 and a drugcontaining one or more free amino groups, wherein the polymer and drugare ionically bonded.
 50. Microparticles according to claim 49 whereinsaid drug is selected from the group consisting of growth hormonereleasing peptide, luteinizing hormone-releasing hormone,adrenomedullin, growth hormone, somatostatin, bombesin, gastrinreleasing peptide, calcitonin, bradykinin, galanin, melanocytestimulating hormone, growth hormone releasing factor, amylin,tachykinins, secretin, parathyroid hormone, erikephalin, endothelin,calcitonin gene releasing peptide, neuromedins, parathyroid hormonerelated protein, glucagon, neurotensin, adrenocorticotrophic hormone,peptide YY, glucagon releasing peptide, vasoactive intestinal peptide,pituitary adenylated cyclase activating peptide, motilin, substance P,neuropeptide Y and TSH or an analogue or a fragment thereof. 51.Microparticles according to claim 50 wherein said drug is somatostatinor LHRH or an analogue or a fragment thereof
 52. Microparticlesaccording to claim 51 wherein said somatostatin analogue isD-β-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-NH₂.
 53. The biodegradable polymeraccording to claim 47 wherein the ratio of lactic acid to glycolic acidto taratic acid is about 66 to about 33 to about 1, respectively. 54.Microparticles comprising a biodegradable polymer according to claim 53.55. Microparticles of a sustained release ionic conjugate comprising thebiodegradable polymer according to claim 53 and a drug containing one ormore free amino groups, wherein the polymer and drug are ionicallybonded.
 56. Microparticles according to claim 55 wherein said drug isselected from the group consisting of growth hormone releasing peptide,luteinizing hormone-releasing hormone, adrenomedullin, growth hormone,somatostatin, bombesin, gastrin releasing peptide, calcitonin,bradykinin, galanin, melanocyte stimulating hormone, growth hormonereleasing factor, amylin, tachykinins, secretin, parathyroid hormone,enkephalin, endothelin, calcitonin gene releasing peptide, neuromedins,parathyroid hormone related protein, glucagon, neurotensin,adrenocorticotrophic hormone, peptide YY, glucagon releasing peptide,vasoactive intestinal peptide, pituitary adenylated cyclase activatingpeptide, motilin, substance P, neuropeptide Y and TSH or an analogue ora fragment thereof.
 57. Microparticles according to claim 56 whereinsaid drug is somatostatin or LHRH or an analogue or a fragment thereof58. Microparticles according to claim 57 wherein said somatostatinanalogue is D-β-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-NH₂.
 59. Thebiodegradable polymer according to claim 47 wherein the ratio of lacticacid to glycolic acid to taratic acid is about 66 to about 32 to about2, respectively.
 60. Microparticles comprising a biodegradable polymeraccording to claim
 59. 61. Microparticles of a sustained release ionicconjugate comprising the biodegradable polymer according to claim 60 anda drug containing one or more free amino groups, wherein the polymer anddrug are ionically bonded.
 62. Microparticles according to claim 61wherein said drug is selected from the group consisting of growthhormone releasing peptide, luteinizing hormone-releasing hormone,adrenomedullin, growth hormone, somatostatin, bombesin, gastrinreleasing peptide, calcitonin, bradykinin, galanin, melanocytestimulating hormone, growth hormone releasing factor, amylin,tachykinins, secretin, parathyroid hormone, enkephalin, endothelin,calcitonin gene releasing peptide, neuromedins, parathyroid hormonerelated protein, glucagon, neurotensin, adrenocorticotrophic hormone,peptide YY, glucagon releasing peptide, vasoactive intestinal peptide,pituitary adenylated cyclase activating peptide, motilin, substance P,neuropeptide Y and TSH or an analogue or a fragment thereof. 63.Microparticles according to claim 62 wherein said drug is somatostatinor LHRH or an analogue or a fragment thereof.
 64. Microparticlesaccording to claim 63 wherein said somatostatin analogue isD-β-Nal-c[Cys-Tyr-D-Trp-Val-Cys]-Thr-NH₂.
 65. A method according toclaim 21 wherein said polyester comprises lactic acid, glycolic acid andtartaric acid.
 66. A method according to claim 28 wherein said polyestercomprises lactic acid, glycolic acid and tartaric acid.
 67. A methodaccording to claim 37 wherein said polyester comprises lactic acid,glycolic acid and tartaric acid.